Process for the disrupting of biological material

ABSTRACT

The present invention relates to a process for the disruption of biological material and to a device for this process. The device according to the invention comprises in combination a pressure vessel provided with inlet and outlet means, pressure regulating means connecting the pressure vessel with a container of a compressed gaseous medium and means for determining the pressure established in the pressure vessel.

United States Patent Schneyour et a1.

[5 PROCESS FOR THE DISRUPIING OF BIOLOGICAL MATERIAL [72] lnventors:Amir Schneyour, 12 a Shalem St., Ramat- Gan; Mordhay Avron, 9 NeveWeizmann, Rehovot, both of Israel [22] Filed: May 15, 1970 [21]Appl.No.: 37,536

[30] Foreign Application Priority Data May 21, 1969 lsrael ..32,268

[52] U.S. Cl. ...241/2 [51] Int. CL... ...-.B02e 19/12 [58] FieldolSearch... ..241/1,2, 18,30

[56] References Cited UNITED STATES PATENTS 1,578,609 3/1926 Mason..241/1 X [451 July 25, 1972 7/1969 Edebe..., ...241/1 1,002,990 9/191 1Herendeen ..241/2 2,928,614 3/1960 Emanuel et a1. ..241/1 X 3,309,0323/1967 Fitz et al. ....241/1 X 2,318,693 5/1943 Joyce et al.... ,.241/l3,165,266 1/1965 -B1um et 241/1 3,556,414 1/1971 Eberly,.lr ..241/2Primary Examiner-Granville Y. Custer, Jr. Attorney-Ostrolenk, Faber,Gerb & Solfen [5 7] ABSTRACT The present invention relates to a processfor the disruption of biological material and to a device for thisprocess. The device according to the invention comprises vin combinationa pressure vessel provided with inlet and outlet means, pressureregulating means connecting the pressure vessel with a container of acompressed gaseous medium and means for determining the pressureestablished in the pressure vessel.

4 Clairm, 1 Drawing Figure BACKGROUND OF THE INVENTION 1. Field of theInvention The invention relates to a process of disrupting biologicalmaterial and to a device for effecting-such process.

2. Description of the Prior Art It is frequently necessary to disruptbiological materiahsuch as bacteria, algae, blood cells, chloroplasts,mitochondria and the like under controlled. and reproducible conditions.In a disruption process, the cell walls are ruptured by firstpressurizing the'cells so'that an equilibrium across the cell walls isattained, andthereafter, suddenly lowering the external pres-- sure,resulting in the cells bursting open due tothe sudden decompression andexpansion of the gas. In the past, rather inconvenient devices were usedfor this purpose. Amongst these, there may be mentioned the French.press and the Hughes press, used in many laboratories. Both devices arerather inconvenient and it is rather laborious to effect the disruptionof the biological material. With the known devices it is hard tomaintain a substantially constant pressure, and it is difficult to carryout the process in a reproducible manner so SUMMARY OF THE PRESENTINVENTION The present invention provides a simple and convenient devicefor accomplishing the foregoing.

The device according to the invention is quite inexpensive and simple.The disruption of the biological material can be effected underaccurately controlled and reproducible conditions, and it can beeffected under any desired gaseous atmosphere.

The process according to the invention comprises introducing thebiological material into a suitable pressure vessel provided with inletand outlet means, establishing above the material a desired. gaseousatmosphere and applying a controlled predetermined pressure byconnecting the pressure vessel via pressure regulating means with ahigh-pressure gas container containing the desired gas, and permittingthe biological material to flow out of the pressure vessel through arelease valve. If necessary, the process may be repeated.

BRIEF DESCRIPTION OF THE DRAWING The invention is described by way ofexample only with reference to the enclosed schematical drawing, whichis a schematic side view of a device according to the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENT The device according to thepresent invention, shown schematically in the enclosed drawing,comprises a pressure vessel (1), provided with a closure (2), an outletvalve (3), an outlet (4), an inlet conduit for the gaseous medium (5),an inlet valve (6), a pressure regulating and pressure indicating valve(7) which is connected via a conduit (8) with a high-pressure gascontainer (9).

For carrying out the process of disrupting biological material accordingto the present invention, the pressure vessel (1) is charged with thebiological material, the atmosphere above the sample is flushed with thegaseous medium (if this is to be other than air), so as to establish adesired gaseous atmosphere above the biological material, the cap (2) isclosed tightly while the valve (6) is in the closed position, thepressure bottle (9) is opened, the pressure is adjusted by means of thevalve (7), the valve 6) is opened so as to establish in the vessel (1)the desired pressure, and subsequently the valve (3) is opened so as togradually eject the biological material through the outlet (4).

The pressure regulating valve 7 makes it possible to establish a desiredgaseous atmosphere of predetermined pres- I exemplified for a number ofbiological systems, without being restricted-thereto.

EXAMPLE 1 A sample of 20 ml of 50 percent w/v Escherichia Coli inphosphate bufier was introduced into a pressure cell and an air pressureof 1,500 p.s.i. was applied; The release was effected'at a, rate of 3 to4 drops per second. After one passage, a very. viscous suspension wasobtaineddue to the liberation of DNA from the disrupted cells. After asecond passage through the pressure cell, in a similar manner, abreakage of 50 percent of the cells was obtained.

In the resulting sample, a protein content of 48mg/ml and a nucleicacids content of 14 mg/ml was obtained. This was determined by opticalmeasurements of a diluted sample.

With a French press, a disruption of percent of the cells can beobtained at a pressure of 15,000 p.s.i., but the biological activity ofthe material is diminished by such high pressures.

EXAMPLE 2 A breakage of 50 percent of the bacterium Rhodospirillumrubrum. was obtained by passage through the device according to theinvention under a pressure of 1,500 p.s.i. of nitrogen. This waseffected in a manner similar to that of Example 1.

By sonication, a breakage of 100 percent was obtained; both of thesebeing determined by the release of bacteric chlorophyll.

The biological activities after disruption by the device according tothe invention were superior as compared with the disruption bysonication or by passage through the French press, as determined bytrans-hydrogenase activity.

Also in the case of Euglena (Example 3), the activity, based ondetermination of phosphorylation activity, was superior in the case ofdisruption by the process according to the invention as compared withsonication.

EXAMPLE 3 The pressure cell according to the invention was chargedEXAMPLE 4 Human red blood cells l-2-percent in saline) were almost 100percent broken by passage through the pressure cell according to theinvention under a pressure of 500 p.s.i. of air.

Comparative runs were made with other compressed gases and'also underincreased pressures. In the latter case, the disruption of subcellularstructures also occurred.

We claim:

1. A process of disrupting biological material, which comprisesintroducing the biological material into a suitable pres sure vesselprovided with inlet and outlet means, establishing above the biologicalmaterial a desired gaseous atmosphere, applying a predetermined pressureon the sample by connecting the pressure vessel with a compressed sourceof said gas, and gradually ejecting the biological material from thepressure vessel through a release valve. 5

2. A process as claimed in claim 1, wherein the applied gas is air,oxygen, nitrogen, argon or helium.

3. A process as claimed in claim 1, wherein the passage through thepressure cell is repeated.

4. A process as claimed in claim 1, wherein the biological material isprovided in the form of a suspension in a suitable suspension medium soas to obtain a liquid suspension of desired viscosity.

* *IK l

1. A process of disrupting biological material, which comprisesintroducing the biological material into a suitable pressure vesselprovided with inlet and outlet means, establishing above the biologicalmaterial a desired gaseous atmosphere, applying A predetermined pressureon the sample by connecting the pressure vessel with a compressed sourceof said gas, and gradually ejecting the biological material from thepressure vessel through a release valve.
 2. A process as claimed inclaim 1, wherein the applied gas is air, oxygen, nitrogen, argon orhelium.
 3. A process as claimed in claim 1, wherein the passage throughthe pressure cell is repeated.
 4. A process as claimed in claim 1,wherein the biological material is provided in the form of a suspensionin a suitable suspension medium so as to obtain a liquid suspension ofdesired viscosity.